These articles have been accepted for publication in the British Journal of Dermatology and are currently being edited and typeset. Readers should note that articles published below have been fully refereed, but have not been through the copy-editing and proof correction process. WileyBlackwell and the British Association of Dermatologists cannot be held responsible for errors or consequences arising from the use of information contained in these articles; nor do the views and opinions expressed necessarily reflect those of Wiley-Blackwell or the British Association of Dermatologists This article is protected by copyright. All rights reserved. Accepted Date : 06-Apr-2014 Article type

: Paediatric dermatology

Propranolol Targets Contractility of Infantile Hemangioma-derived Pericytes

D. Lee1, E. Boscolo1,2, J.T. Durham3, J.B. Mulliken2,4, I.M. Herman3, J. Bischoff 1,2 Affiliations: 1Vascular Biology Program and Department of Surgery, Boston Children’s Hospital, Boston, MA; 2Department of Surgery, Harvard Medical School, Boston, MA; 3Graduate Program in Cellular and Molecular Physiology, Sackler School of Graduate Biomedical Sciences and Department of Integrative Physiology and Pathology, School of Medicine, Tufts University, Boston, MA; 4Department of Plastic and Oral Surgery, Boston Children’s Hospital Boston, MA Running head: Propranolol targets contractility of hemangioma pericytes Corresponding Author:

Joyce Bischoff, PhD Boston Children’s Hospital 300 Longwood Ave, Boston, MA 02115 Phone: 617-919-2192 Fax: 617-730-0231 E-mail: [email protected]

Funding: National Institutes of Health, Award Numbers P01 AR048564 and HL 096384 (J.B.), Charles H. Hood Foundation (E.B.) Conflicts of interest: none What is already known about this topic? Propranolol is an effective treatment for problematic infantile hemangioma.

This article is protected by copyright. All rights reserved. Propranolol, an antagonist of β-adrenergic receptors, reduces proliferation and increases apoptosis of hemangioma endothelial cells in vitro. What does this study add? Propranonol increased contractility of hemangioma pericytes but had no effect on normal pericytes. Propranolol significantly reduced the vascular volume of blood vessels assembled from hemangioma pericytes and endothelial cells. Hemangioma pericytes are a likely target of propranolol.

Abstract: Propranolol, a β-adrenergic receptor (AR) antagonist, was discovered serendipitously to be an effective treatment for endangering infantile hemangioma (IH). Dramatic fading of cutaneous color is often seen a short time after initiating propranolol therapy, with accelerated regression of IH blood vessels discerned after weeks to months. Here we focus on hemangioma-derived pericytes (HemPericytes) isolated from proliferating and involuting phase tumors to assess a possible role for these cells in the apparent propranolol-induced vasoconstriction. HemPericytes express high levels of β2 AR mRNA, compared to positive control bladder smooth muscle cells. In addition, β2 AR mRNA levels were relatively high in IH specimens (n=15) compared to β1 AR, β3 AR and α1bAR. HemPericytes were assayed for contractility on a deformable silicone substrate: propranolol (10µM) restored basal contractile levels in HemPericytes that were relaxed with the AR agonist epinephrine. siRNA knockdown β2 AR blunted this response. Normal human retinal and placental pericytes were not affected by epinephrine or propranolol in this assay. Propranolol (10µM) inhibited proliferation of HemPericytes in vitro, as well as normal pericytes, indicating a non-selective effect in this assay. HemPericytes and HemEC were co-implanted subcutaneously in nude mice to form blood vessels, and at day 7 after injection, mice were randomized into vehicle and propranolol treated groups. Contrast-enhanced micro-ultrasonography of the implants after 7 days of treatment showed significantly decreased vascular volume in propranolol-treated animals, but no reduction in vehicle-treated animals. These findings suggest that the mechanism of propranolol’s effect on proliferating IH involves increased pericytic contractility.

This article is protected by copyright. All rights reserved. Introduction: Infantile hemangioma (IH) is a benign vascular tumor that occurs in 4-5% of infants 1. The tumor follows a unique life-cycle of rapid growth, called the proliferating phase, followed by a slow spontaneous involuting phase. This sequence of abnormal vascular growth followed by regression has long fascinated researchers in the field of angiogenesis as a potential wellspring for clues to the regulation of human vascular development and post-natal angiogenesis. For most children, IH does not pose a serious threat and therapy is unnecessary; however, in some instances, IH can enlarge dramatically, threaten vital organs and cause permanent disfigurement. Corticosteroid therapy had been the mainstay treatment until the discovery that oral propranolol accelerates the regression of IH 2. Within a relatively short period of time, propranolol has replaced corticosteroid as the first-line therapy for problematic IH. Knowledge of the cellular and molecular mechanisms by which propranolol exerts its effect might lead to further improvements to shorten the duration of treatment, prevent the rebound growth that occurs in some cases 3 and help understand why some IH do not respond to propranolol therapy 4. Storch and Hoeger reviewed three broad mechanisms by which propranolol might act on IH 5. First, propranolol might antagonize β-AR-mediated vasodilation, leading to vasoconstriction of IH vessels, which would explain the rapid reduction in volume and color seen with propranolol therapy 6. Second, propranolol might block VEGF-mediated angiogenesis, since β-AR signaling has been shown to increase VEGF-A expression 7. Third, propranolol might increase apoptosis, as β-AR agonists have been shown to inhibit apoptosis in many different cell types5. This would be consistent with the accelerated onset of involution observed in IH patients on propranolol therapy. In support of these mechanisms, β-ARs have been detected in IH tumor specimens 8,9 10 and hemangioma-derived endothelial cells (HemEC) and hemangioma-derived stem cells (HemSC) 10-12 . These reports, combined with data in Figure 1, document the presence of the critical receptor targets for propranolol in IH, establishing a basis for functional studies. Two groups showed that high concentrations of propranolol (100-300µM) reduced VEGF-A levels in HemEC13,14. Chim et. al. also found that propranolol reduced hypoxia inducible factor1α and matrix metalloproteinase-2 13. Ji and colleagues reported that propranolol decreased proliferation, induced cleaved caspase-9 and caspase-3 and increased pro-apoptotic proteins p53 and Bax in HemEC 9. Wong and coworkers confirmed the increased apoptosis 14, but other investigators found no increases in apoptosis of HemEC treated with propranolol 15. In a later study, Ji and colleagues showed that a β2-selective antagonist ICI 118551 (10µM) was more effective than the β1-selective antagonist metaprolol (10µM) in blocking β-AR agonist-

This article is protected by copyright. All rights reserved. stimulated HemEC proliferation 11, suggesting an important role for β2 AR in this context. Of note, many reports on IH-derived cells studied effects of propranolol in the absence of added agonists. Thus, propranolol may be active as an inverse agonist, that is, inhibiting basal levels of β-AR signaling, as has been shown in other settings 16,17. We previously showed that HemPericytes from proliferating and involuting phase IH have a reduced contractile activity in vitro and are pro-angiogenic compared to normal human pericytes 18. Herein, we analyzed: 1) expression of β-ARs in HemPericytes, 2) contractile activity of HemPericytes in response to propranolol in the presence/absence of the β-AR agonist epinephrine, 3) proliferation of HemPericytes in presence of propranolol and 4) effect of a one week propranonol treatment on blood vessels assembled from HemPericytes and HemEC in mice. Our findings indicate that propranolol reverses epinephrine-induced relaxation of HemPericytes in vitro and reduces the vascular volume of HemPericyte/HemECderived vessels in vivo. The propranolol-mediated increased contractility of hemangioma pericytes could explain the apparent vasoconstriction of IH vessels after the initiation of propranolol therapy. Materials and Methods In vitro culture of HemPericytes, HemEC and HemSC Specimens of IH were obtained under a protocol approved by the Committee on Clinical Investigation, Boston Children’s Hospital. The clinical diagnosis was confirmed by histopathology. Hem-Pericytes, HemEC and HemSC were isolated as described 18-20. HemPericytes were cultured on non-coated tissue culture plates in DMEM 10% fetal bovine serum (FBS), 1x glutamine-penicillin-streptomycin (GPS)18. HemEC and HemSC were cultured on fibronectin-coated (1 µg/cm2) plates in endothelial basal medium (EBM-2, Lonza CC-3156) supplemented with 20% FBS, EGM-2 SingleQuots without hydrocortisone (Lonza CC-4176) and 1x GPS20. Non-hemangioma cell culture Human dermal microvascular endothelial cells (HDMEC) were isolated from neonatal foreskin21 and cultured as described for HemEC and HemSC. Human placental and retinal pericytes were purchased from Promocell (C-12981) and Cell Systems (ACBRI 183), respectively, and grown as described for HemPericytes. Smooth muscle cells (SMC) from bladder and bronchus were purchased from ScienCell and cultured in SMC media from ScienCell (Cat. 1101).

Quantitative real-time polymerase chain reaction (qRT-PCR) RNA from cells and tissues was extracted using RNeasy Micro kit (Qiagen) and TRIzol (Invitrogen), respectively. cDNAs were synthesized with Quanta qScript cDNA Synthesis kit

This article is protected by copyright. All rights reserved. (VWR Scientific). Primers for qRT-PCR are shown in Table 1. Four housekeeping genes, GAPDH, β-actin, HGPRT, and ribosomal protein S9 were evaluated for consistency among samples: GAPDH was selected for normalizing IH tissue RNA and β-actin was selected for normalizing RNA isolated from IH-derived cells. Reactions were prepared with SYBR Green master mix (Roche) and performed on Applied Biosystems’ StepOnePlus Real-Time PCR system. Contractility assay A deformable silicone-coated substratum was prepared as described 22,23. Polydimethylsiloxane from Sigma-Aldrich (DMPS12M-100G, viscosity 12,500 cST) was pipetted onto marked, round glass coverslips (Fisher Scientific 12-545-81) and spread for one hour at 40 °C, then thermally cross-linked and coated with electrical charge for 13 seconds using a plasma etcher (SPI PlasmaPrep II 11005). The coverslips were coated with 0.1 mg/mL of rat type I collagen (BD Biosciences) and placed in 24-well plates. Plates were UV-irradiated for five minutes and 4000 pericytes in DMEM 2% FBS, 1x GPS were pipetted into each well. This assay tested HemPericytes from three different proliferating phase IH (146, 154 and 156), three different involuting phase IH (I-69, I-79 and I-82) and normal human pericytes from retina and placenta. After 48 hours, images were taken under bright field at 10X (Nikon Eclipse TS100) to visualize and quantify “wrinkled” contractile cells at time=0. Contractility at time = 0 was set to 100%. In a blinded fashion, cells were treated with vehicle (n = 6 wells), propranolol (10 µM) (n = 6 wells), vehicle + epinephrine (5.5 µM) (n = 6 wells) or propranolol + epinephrine (n = 6 wells). Propranolol was from Sandoz (NDC 0781-3777-95, 1mg/ml, pH 4), epinephrine was from Hospira (NDC 0409-7241-01, 1 mg/mL) and the vehicle was prepared by the Boston Children’s Hospital Pharmacy to match the solution in which propranolol was supplied. One hour after addition of reagents, wrinkled cells were located and images taken. Contractility was quantitated in all four conditions using the formula: C = N x L, N = # of wrinkles and L = length in pixels from ImageJ 24. siRNA silencing of β2 AR in HemPericytes Non-targeting siRNA and β2-AR siRNAs were purchased from Dharmacon (D-001810-03-05 and L-005426-01-0005). Non-targeting siRNA has no homology to any known human gene. β2-AR siRNAs were a pool of 5 siRNAs, designed to maximize knockdown. HemPericytes from proliferating phase IH (146, 154 and 156) and involuting phase IH (I-69, I-79 and I-82) were grown in DMEM 10% FBS without antibiotics until they reached 60-90% confluence on 12-well plates. Cells were transfected with 10 nM of siRNA using Lipofectamine RNAiMAX (Life Technologies), prepared in reduced-serum media (Gibco OPTI-MEM). The cells were trypsinized 24 hours after transfection and prepared for contractility assays or re-plated on 12well plates to assess extent of β2-AR of knockdown by qRT-PCR. The contractility assays and qRT-PCR were performed 48 hours after transfection.

This article is protected by copyright. All rights reserved. Proliferation Assays Cells were plated at 5000 cells/cm2 in 48-well plates in growth medium (n=4/condition). Cells were treated twice daily with PBS or propranolol (10 µM) from Day 1 to 5. Cells were counted using an automated cell counter (Millipore Scepter 2.0); all particles whose diameters ranged 10-25 µM were included. HemSC isolated from three different IH (94, 97, 133), HemEC isolated from three different IH (150, 158, I-69), and HemPericytes from three different IH (146, 154, 156) were assayed. In vivo study and micro-ultrasonography analysis HemECs (158) and HemPericytes (154), 1.25 x 106 of each cell type, were suspended in 250 µl of Matrigel and injected subcutaneously on the backs of 20 6-week old male athymic nude/nude mice20. Two additional mice were injected with 250 µl of Matrigel without cells and served as negative controls. At day 7, micro-ultrasonography was used to assess perfused vascular volume25,26. Mice with HemPericyte/HemEC implants with contrast values above those of negative controls were treated with vehicle or propranolol at 5 mg/kg, twice a day for 7 days, by i.p. injection. Vascular volume was determined by contrast-enhanced micro-ultrasonography using a VisualSonic Vevo 2100 as described25,26. Tail veins of mice were cannulated using modified, winged infusion set (Allegro Medical 549230); non-targeted contrast agent (VisualSonics VS11913) was injected to quantify vascular volume in the implants. Twenty minutes passed for contrast agent to be cleared from circulation and the above steps were repeated. The two measurements were averaged to determine contrast values. Microvascular density (MVD) and immunostaining for human CD31+ vessels Twenty images from mid-Matrigel H & E sections of all animals in vehicle and propranolol groups were taken randomly at 40X (Zeiss Axiophot II, equipped with AxioCam MRc5 and supplemented with AxioVision Rel. 4.8 software). Luminal structures containing at least one RBC were counted as one vessel; the average from 20 images was expressed as vessels/mm2. Formalin-fixed, paraffin-embedded mid-Matrigel sections were processed and stained with mouse anti-human CD31 (1:40, Dako M0823) or mouse IgG (1:40, Dako X0931) as described18. Statistical analyses Contractility assays were analyzed by two-tailed, paired t-tests. Cellular proliferation assays and histological analyses were analyzed by two-tailed, unpaired t-tests. Contrast values of mice injected with HemECs 158 and Hem-Pericytes 154 were analyzed by two-tailed, paired t-tests. All data analyses were performed using GraphPad Prism Version 5.04 and differences were considered significant when p < 0.05.

This article is protected by copyright. All rights reserved. Results β1-AR and β2-AR mRNA in hemangioma-derived cells and hemangioma tissue. β1-AR and β2-AR mRNA transcript levels were quantified in HemEC, HemPericytes and HemSC isolated from different IH specimens. β1-AR was detected at low levels in HemEC but was undetectable in HemPericytes and HemSC (Figure 1A). β2-AR was detected in HemEC and HemPericytes at levels above or comparable to bladder smooth muscle cells, the positive control. HemSC expressed low but detectable levels of β2-AR (Figure 1B). We extended this analysis to IH specimens from 15 patients at ages ranging from 3 months to 10 years. β2-AR was prominently expressed in most IHs, with the exception of 2: one from a 15 month old and one from a 21 month old child (Figure 1D). These two specimens were shown to have expected levels of the endothelial marker CD31 and the pericyte/smooth muscle marker α-smooth muscle actin (data not shown). β1-AR, β3-AR and the α1b-AR were detected at low and variable levels in IH specimens (Figure 1C, E, F). In summary, mRNA transcript levels for these four AR were variable among the 15 IH specimens tested. Propranolol reverses epinephrine-induced relaxation of HemPericytes Pericytes are contractile and regulate capillary blood flow. In vitro, calf serum stimulates pericytic contractility while β-AR agonists and cAMP analogs cause pericytic relaxation 14,27. We previously reported that HemPerictyes exhibit reduced contractile activity in vitro compared to normal human retinal and placental pericytes18. Therefore, we examined contractility of HemPericytes and normal pericytes in response to propranolol and a β-AR agonist epinephrine. Pericytes were plated on type I collagen-coated silicone membranes and allowed to “wrinkle” the silicone substratum, indicating contractility (Figure 2A). Wrinkled pericytes were quantified at 48 hours, t= 0, and this value was set at 100%. Propranolol and/or epinephrine were added as indicated for one hour and wrinkled cells were re-quantified. Propranonol was tested at 10 µM based on prior studies in which 10 µM propranolol was sufficient to completely block norepinephrine-induced dilation of human coronary arterial segments 28. The percent change in contractility compared to t=0 in HemPericytes from three different proliferating phase IH, three different involuting phase IH, and normal human pericytes is shown in Figure 2B. Epinephrine-induced relaxation of proliferating HemPericytes was prevented by propranolol (35.4 ±42.1 versus 89.6 ±6.9 contractile cells; p=0.018). Epineprhine-induced relaxation of involuting HemPericytes was also prevented by propranolol (49.3 ± 26.3 versus 73.3 ± 25.2; p= 0.021). Normal human pericytes from retina and placenta did not relax in response to epinephrine and there was no detectable effect of propranolol (89.2± 11 versus 98.6 ± 7.2; p= 0.247)(Figure 2B). Supplemental Figure 2 shows individual contractility data for each pericyte

This article is protected by copyright. All rights reserved. population - hemangioma-derived and normal. Supplemental Figure 1 shows β2-AR mRNA levels, determined by qRT-PCR, for each of the pericytes analyzed in the contractility assay. We used siRNA silencing to reduce β2-AR expression in all HemPericytes (Figure 2C), which resulted in a loss of epinephrine-induced relaxation, and thus a loss of the propranololmediated reversal of this relaxation (Figure 2C). This data strongly implicates β2-AR to be involved in relaxation and propranolol-mediated contractility of HemPericytes in this in vitro assay. The extent to which β2-AR mRNA levels were reduced by siRNA is shown by qRT-PCR (Supplemental Figure 2). Propranolol inhibits proliferation of HemPericytes and normal pericytes Proliferation of HemPericytes 154, in the presence of 10µM propranolol added twice daily over 4 days, was inhibited by 41% compared to the control (Figure 3A). Propranonol reduced the proliferation of placental and retinal pericytes by 48% and 59%, respectively. For comparison, we also assayed the proliferation of HemSC-133, HemEC-158 and HDMEC. Propranolol did not affect the proliferation of HemSC or HDMEC, although it reduced proliferation of HemEC-158 by 37%. The results of proliferation assays of hemangioma-derived cells from three different IH tumors were combined: HemPericytes (n=3), HemEC (n=3) and HemSC (n=3) (Figure 3C). This confirmed the statistically significant inhibitory effect of propranolol on HemPericyte and HemEC proliferation. The results confirm previous studies on the inhibitory effect of propranolol on HemEC 12,14 and show that proliferation of HemPericytes is also inhibited by propranolol, which may contribute to the anti-angiogenic effect on IH. Propranolol reduces vascular volume in blood vessels formed in vivo by implanting HemPericytes and HemEC. We previously showed that HemPericytes co-implanted with normal human EC or HemEC formed perfused vascular networks in immune-deficient mice 18. Here, we co-implanted HemPericytes + HemEC and allowed vascular networks to form until Day 7, at which time vascular volume in the implants was measured using contrast-enhanced microultrasonography. The mice were divided into two groups and treated twice daily for 7 days with either vehicle or propranolol by i.p. injection. Vascular volume was re-assessed on Day 14. Heart rates were 390 ± 52 for the vehicle treated group and 413±5 for the propranolol treated group. Contrast values for individual animals and mean contrast values at Day 7 and Day 14 are shown in Figure 4. Mice treated with propranolol showed a significant reduction in mean contrast value at Day 14, whereas mice treated with vehicle did not. It is unlikely that the propranolol effect was due to changes in blood pressure because in similar experiments in

This article is protected by copyright. All rights reserved. which HemSC were implanted as a single cell type, we did not see a statistically significant reduction in vascular volume in propranolol-treated mice (data not shown). Propranolol had no significant effect on the number of vessels in the implants at Day 14, as determined by counting the number of red blood cell-filled vessels (Figure 4C). To verify that red blood cellfilled vessels were composed of human endothelial cells, sections were stained with an antihuman CD31 mAb (Figure 4D). In this assay, propranolol did not reduce the number of vessels but reduced the perfused vascular volume, consistent with the phenomenon of increased vasoconstriction. Discussion Hemangioma-derived endothelial cells (HemEC) and stem cells (HemSC) have been shown to be variably affected by propranolol in in vitro assays for proliferation, differentiation and apoptosis. In contrast, relatively little is known about pericytes in IH and how these cells might respond to propranolol. We show that HemPericytes express β2AR mRNA and that propranolol reverses epinephrine-induced relaxation of HemPericytes. A limitation of our study is that we did not analyze β2AR at the protein level. In vivo, propranolol treatment for 7 days reduced perfused vascular volume of blood vessel networks formed from HemPericytes and HemEC. These findings indicate that propranolol increases the contractile activity of HemPericytes, which may contribute to vasoconstriction and reduced blood flow in IH. HemPericytes isolated from different proliferating phase IH specimens were found to exhibit reduced contractility and increased proliferation compared to normal human pericytes from human retina or placenta18. Propranolol reduced proliferation of HemPericytes and HemEC, indicating that the agent may also have an anti-angiogenic effect in vivo. Propranolol did not inhibit proliferation of fast-growing vasculogenic HemSC that are able to differentiate into pericytes and endothelial cells, and form IH-like vessels in immune-deficient mice20,29. Considered together with published reports, our data indicate propranolol can target endothelial cells and pericytes in IH, exerting overlapping and distinctive effects on each. More studies are needed to determine how these in vitro studies correlate with clinical response to propranolol therapy. In rare instances, some children with IH fail to respond to propranonol therapy. Perhaps low or undetectable levels of β1 and β2-AR, as seen in some IH specimens (Figure 1C,D), explain the ineffective propranolol therapy in these uncommon cases. Analyses of β-AR expression in IH specimens from patients that do not respond to propranolol would be needed to pursue this hypothesis. Another un-explained feature of propranolol therapy is the clinical regrowth of IH that occurs in approximately 20% of cases when the drug is discontinued 3 . This observation suggests that propranolol does not produce irreversible effects, either antiproliferative or anti-vasculogenic, on the cells that drive hemangioma-genesis. Further studies

This article is protected by copyright. All rights reserved. are needed to decipher the clinical features of the rebound phenomenon, combined with in vitro and in vivo experiments, in order to investigate these concepts.

Acknowledgements – Research reported in this manuscript was supported by the NHLBI and NIAMS of the National Institutes of Health under award number R01 HL096384 and P01 AR048564 (J.B.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Funding for this research was also from the Charles H. Hood Foundation (E.B.). We thank Dr. David Zurakowski, Boston Children’s Hospital for advice on statistical analyses. Figure Legends Figure 1 – β-AR mRNA in hemangioma-derived cells and hemangioma tissue Panels A-B, mRNA levels in HemEC (n= 7), HemPericytes (n=3) and HemSC (n=8) quantified by qRT-PCR. A, β1-AR mRNA in human cardiac tissue, normalized to GAPDH, set = 1.0. Relative levels in cells, normalized to GAPDH, are shown. B, β2-AR mRNA in human bladder SMC, normalized to GAPDH set = 1.0. Relative levels in cells, normalized to GAPDH, are shown. Panels C-F, IH specimens removed from patients at different ages (shown in months or years) analyzed for C, β1-AR, D, β2-AR, E, β3-AR, and F, α1bAR mRNA levels by qRT-PCR. β3-AR mRNA in human foreskin, normalized to β-actin, set = 1.0. α1bAR mRNA in bronchial SMC, normalized to β-actin, set = 1.0. Relative levels in tissue, normalized to β-actin, are shown. Figure 2 – Propranonol effects on HemPericyte and normal pericyte contractility A, Diagram of contractility assay with phase image of HemPericyte wrinkling the silicone substratum. B, Contractility of proliferating phase HemPericytes (white bars), involuting phase HemPericytes (grey bars) and normal human pericytes (dark grey bars) at 48 hours after plating was quantified and set to 100%. Percent change in contractility was determined after cells were treated for one hour with vehicle, propranolol , vehicle + epinephrine or propranolol + epinephrine . C, Contractility assays performed on HemPericytes (black bars) with corresponding siRNA β2-AR knockdown (grey bars). Values are mean ± standard deviation. Data analyzed by two-tailed, paired t-tests. Figure 3 – Propranolol effects on proliferation of HemPericytes, normal pericytes, HemEC and HemSC A, Representative proliferation assay with HemPericyte 154, placental pericytes and retinal pericytes and in B, HemSC-133, HemEC-158 and normal human endothelial cells (HDMEC). Cells plated at 5000 cells/cm2 in growth media in 48 well dishes (n=4/condition). Cells allowed to adhere for 24 hours (Day 1), and then treated twice daily with PBS (white bars) or propranolol (10µM) (gray bars) until Day 5. Cells counted on Day 1 and Day 5. Values are mean ± standard deviation. Data analyzed by two-tailed, unpaired t-tests. C, Proliferation at

This article is protected by copyright. All rights reserved. Day 5 in PBS set to 100% (white bars) versus propranonlol (grey bars) of HemPericytes 146, 154, 156; HemEC 150, 158, I-69 and HemSC 94, 97, 133. Figure 4 – Propranolol effects on vascular volume in HemPericyte/HemEC Matrigel implants in immune-deficient mice. Immune-deficient nude mice (n=20) implanted sub-cutaneously with HemPericytes and HemEC suspended in Matrigel. At Day 7, mice treated twice daily with vehicle or propranolol by i.p. injection for 7 days. Vascular volume determined by contrastenhanced micro-ultrasonography. A, Contrast values for individual mice. B, Mean contrast values at Day 7 and Day 14; data analyzed by two-tailed, paired t-tests. C, Vessel density at Day 14 and D, Control IgG and anti-human CD31 immunostaining of sections from implants from vehicle-treated and propranolol-treated mice at Day 14.

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Boscolo E, Stewart CL, Greenberger S et al. JAGGED1 signaling regulates hemangioma stem cell-to-pericyte/vascular smooth muscle cell differentiation. Arterioscler Thromb Vasc Biol 2011; 31: 2181-92.

Table 1. Sequences of primers used for qRT-PCR.

Gene

Forward

Reverse

α1b-AR

5’-GATCCATTCCAAGAACTTTCAC-3’

5’-CAGAACACCACCTTGAACAC-3’

β1-AR

5’-TCTTTTGTGTGTGCGTGTGA-3’

5’-ATGCTTCTCCCTTCCCCTAA-3’

β2-AR

5’-CACCAACTACTTCATCACTTCAC-3’

5’-GACACAATCCACACCATCAG-3’

β3-AR

5’-TGAAATCCAGTTGCCATTGA-3’

5’-CACCATGTAAGGCACCACTG-3’

β-actin

5’-TGAAGTGTGACGTGGACATC-3’

5’-GGAGGAGCAATGATCTTGAT-3’

GAPDH

5’-TGCACCACCAACTGCTTAG-3’

5’-GATGCAGGGATGATGTTC-3’

HPRT1

5’- TGGACAGGACTGAACGTCTTG-3’

5’- CCAGCAGGTCAGCAAAGAATTTA-3’

S9

5’-GATTACATCCTGGGCCTGAA-3’

5’-ATGAAGGACGGGATGTTCAC-3’

This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.